TOF-SIMS analysis of the interface between bone and titanium implants—Effect of porosity and magnesium coating

Implant healing was studied with regard to the mineralization of the implant-tissue interface. Titanium discs were surface-modified and implanted in rat tibia for 4 weeks. After implantation, the bone was embedded in resin and cross sections of bone and implant were made using a low speed saw equipped with a diamond wafering blade. The sections were analyzed with imaging TOF-SIMS using a Bi₃⁺ cluster ion source. This ion source has recently been shown to enable identification of hydroxyapatite (HA) fragments in bone samples. The area within 40 μm from the implant surface was selected for analysis, corresponding to bone-implant interface, from which positive spectra were recorded. In conclusion, differences were observed between the implants tested regarding signal intensity of fragments specific for HA. Coating of the implants with magnesium and porosity were shown to influence the mineral content of the bone-implant interface. This technique might be useful for biocompatibility assessment and for studying the mineralization process at implant surfaces.     

Localization of Na and K in rat cerebellum with imaging TOF-SIMS

High pressure-frozen (HPF), freeze-fractured and freeze-dried rat cerebellum was analyzed with imaging TOF-SIMS equipped with a Bi-cluster ion source. Data were collected separately as spectra of high mass resolution m/Δm > 8000 and images of high lateral resolution <700 nm. Images were made showing the localization of the peaks m/z = 22.99, and m/z = 39.1.Topographical effects were noted due to the freeze fracture method. This effect was compensated by normalizing images of specific secondary ions to the intensity of total secondary ions and by making PCA analysis of the image. The results showed that potassium ions were localized in blood vessels and cortex cells and sodium ions were localized in blood vessels and cerebellar interstitial tissue and in the nuclei of some cells. The sodium ion concentration was found to be higher in blood vessels than in the interstitium.     

Molecular imaging of enhanced Na expression in the liver of total sleep deprived rats by TOF-SIMS

Sleep disorder is associated with metabolic disturbances, which was related to oxidative stress and subsequently sodium overload. Since liver plays important roles in metabolic regulation, present study is aimed to determine whether hepatic sodium, together with oxidative stress, would significantly alter after total sleep deprivation (TSD). Sodium ion was investigated by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Parameter for oxidative stress was examined by heat shock protein-25 (HSP-25) immunohistochemistry. TOF-SIMS spectrum indicated that hepatic Na⁺/K⁺ ratio counting as 82.41 ± 9.5 was obtained in normal rats. Sodium ions were distributed in hepatocytes with several aggregations. However, following TSD, the intensity for Na⁺/K⁺ ratio was relatively increased (101.94 ± 6.9) and signals for sodium image were strongly expressed throughout hepatocytes without spatial localization. Quantitative analysis revealed that HSP-25 staining intensity is 1.78 ± 0.27 in TSD rats, which was significantly higher than that of normal ones (0.68 ± 0.15). HSP-25 augmentation suggests that hepatocytes suffer from oxidative stress following TSD. Concerning oxidative stress induced sodium overload would impair metabolic function; enhanced hepatic sodium expression after TSD may be a major cause of TSD relevant metabolic diseases.     

Deposition of titanium nitride and hydroxyapatite-based biocompatible composite by reactive plasma spraying

Titanium nitride is a bioceramic material successfully used for covering medical implants due to the high hardness meaning good wear resistance. Hydroxyapatite is a bioactive ceramic that contributes to the restoration of bone tissue, which together with titanium nitride may contribute to obtaining a superior composite in terms of mechanical and bone tissue interaction matters.The paper presents the experimental results in obtaining composite layers of titanium nitride and hydroxyapatite by reactive plasma spraying in ambient atmosphere. X-ray diffraction analysis shows that in both cases of powders mixtures used (10% HA + 90% Ti; 25% HA + 75% Ti), hydroxyapatite decomposition occurred; in variant 1 the decomposition is higher compared with the second variant. Microstructure of the deposited layers was investigated using scanning electron microscope, the surfaces presenting a lamellar morphology without defects such as cracks or microcracks. Surface roughness values obtained vary as function of the spraying distance, presenting higher values at lower thermal spraying distances.     

Surface characterisation and electrochemical behaviour of porous titanium dioxide coated 316L stainless steel for orthopaedic applications

Porous titanium dioxide was coated on surgical grade 316L stainless steel (SS) and its role on the corrosion protection and enhanced biocompatibility of the materials was studied. X-ray diffraction analysis (XRD), atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX) were carried out to characterise the surface morphology and also to understand the structure of the as synthesised coating on the substrates. The corrosion behaviour of titanium dioxide coated samples in simulated body fluid was evaluated using polarisation and impedance spectroscopy studies. The results reveal that the titanium dioxide coated 316L SS exhibit a higher corrosion resistance than the uncoated 316L SS. The titanium dioxide coated surface is porous, uniform and also it acts as a barrier layer to metallic substrate and the porous titanium dioxide coating induces the formation of hydroxyapatite layer on the metal surface.     

Advances in the ultrastructural study of the implant-bone interface by backscattered electron imaging

The biocompatibility of titanium implants in bone depends on the response shown by cells in contact with the implant surface. Several developments have been targeted at achieving successful implant treatment. The aim of this study was to develop a novel preparation procedure to evaluate the bone cell response produced at the bone–implant interface using the technique scanning electron microscopy with backscattered electron imaging (SEM-BSE). Dental prostheses with an SLA-modified or TOP-modified surface were implanted in a toothless part of the mandibula in female pigs. The animals were sacrificed 12 weeks after surgery, at which time block specimens containing the implants were obtained. These specimens were then processed for SEM-BSE by optimizing a protocol involving chemical fixation and heavy metal staining. In addition, element distribution maps for the implant–bone tissue interface were obtained using a microanalytical system based on energy-dispersive X-ray spectrometry (EDS). This novel visualisation approach enabled a comprehensive study of the extracellular matrix and cell components of the host tissues neoformed around the implant. SEM-BSE images also provided ultrastructural details of the bone cells. This technique appears to be an effective and very promising tool for detailed studies on the implant–bone tissue interface and the host response to the bone incorporation process.     

Composite coating of 58S bioglass and hydroxyapatite on a poly (ethylene terepthalate) artificial ligament graft for the graft osseointegration in a bone tunnel

The purpose of this study was to determine the effect of the combination of hydroxyapatite (HA) and bioglass (BG) on polyethylene terephthalate (PET) artificial ligament graft osseointegration within the bone tunnel. The results of in vitro culturing of MC3T3-E1 mouse osteoblastic cells proved that this HA/BG composite coating can promote the cell compatibility of grafts. A rabbit extraarticular tendon-to-bone healing model was used to evaluate the effect of this composite coating on PET artificial ligaments in vivo. The final results demonstrated that HA/BG coating improved new bone formation at the graft-bone interface and increased the load-to-failure property of graft in bone tunnel compared to the control group at early time. The study has shown that HA/BG composite coating on the PET artificial ligament surface has a positive effect in the induction of artificial ligament osseointegration within the bone tunnel.     

Selective detection of organic compounds on modified polymer surfaces using TOF-SIMS in combination with derivatization

This investigation encompasses work in the development of TOF-SIMS methodology for the characterization of compounds formed during polymer surface modification. TOF-SIMS was used in this study in combination with selective derivatization reactions with hydroxyl group specific reagents. Derivatization techniques with group specific reagents provide a means of identifying functional groups in a complex matrix, along with significant enhancement of detection limits. The study proceeded in three steps. First, derivatives of organic compounds as a model compound were monitored to determine the suitability for detecting oxygenated species. Second, useful derivatization reactions were tested on functional groups in the synthetic polymer chains. Third, the methods thus developed were applied to polymer surfaces treated by plasma source ion implantation (PSII). 2-Fluoro-1-methylpyridinium derivatives were useful for characterization of organic alcohols and phenolic compounds. Analysis of organic compounds derivatized by these methods on polymeric materials demonstrated clearly that analysis in the presence of a carbonaceous matrix is possible. The results yielded evidence for the formation of hydroxyl species as the polymer is modified by PSII technique.     

Bioactive coating on titanium implants modified by Nd:YVO4 laser

Apatite coating was applied on titanium surfaces modified by Nd:YVO₄ laser ablations with different energy densities (fluency) at ambient pressure and atmosphere. The apatites were deposited by biomimetic method using a simulated body fluid solution that simulates the salt concentration of bodily fluids. The titanium surfaces submitted to the fast melting and solidification processes (ablation) were immersed in the simulated body fluid solution for four days. The samples were divided into two groups, one underwent heat treatment at 600 °C and the other dried at 37 °C. For the samples treated thermally the diffractograms showed the formation of a phase mixture, with the presence of the hydroxyapatite, tricalcium phosphate, calcium deficient hydroxyapatite, carbonated hydroxyapatite and octacalcium phosphate phases. For the samples dried only the formation of the octacalcium phosphate and hydroxyapatite phases was verified. The infrared spectra show bands relative to chemical bonds confirmed by the diffraction analyses. The coating of both the samples with and without heat treatment present dense morphology and made up of a clustering of spherical particles ranging from 5 to 20 μm. Based on the results we infer that the modification of implant surfaces employing laser ablations leads to the formation of oxides that help the formation of hydroxyapatite without the need of a heat treatment.     

Up-regulation of Na expression in the area postrema of total sleep deprived rats by TOF-SIMS analysis

Area postrema (AP) is a circumventricular organ plays an important role in sodium homeostasis and cardiovascular regulation. Since sleep deficiency will cause cardiovascular dysfunction, the present study aims to determine whether sodium level would significantly alter in AP following total sleep deprivation (TSD). Sodium level was investigated in vivo by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Clinical manifestation of cardiovascular function was demonstrated by mean arterial pressure (MAP) values. Results indicated that in normal rats, TOF-SIMS spectrum revealed a major peak of sodium ion counting as 5.61 × 10⁵ at m/z 23. The sodium ions were homogeneous distributed in AP without specific localization. However, following TSD, the sodium intensity was relatively increased (6.73 × 10⁵) and the signal for sodium image was strongly expressed throughout AP with definite spatial distribution. MAP of TSD rats is 138 ± 5 mmHg, which is significantly higher than that of normal ones (121 ± 3 mmHg). Regarding AP is an important area for sodium sensation and development of hypernatremic related sympatho-excitation; up-regulation of sodium expression following TSD suggests that high sodium level might over-activate AP, through complex neuronal networks involving in sympathetic regulation, which could lead to the formation of TSD relevant cardiovascular diseases.     

Preparation and antibacterial effect of silver-hydroxyapatite/titania nanocomposite thin film on titanium

The composite which contains Ag⁺ and nanosized hydroxyapatite with TiO₂ was deposited onto titanium by dipping method. The morphology, chemical components and structures of the thin film were characterized by XRD, scanning electronic microscope (SEM) and energy dispersive X-ray analysis (EDX). Staphylococcus aureus and Escherichia coli were utilized to test the antibacterial effect. XRD results demonstrated that the films have characteristic diffraction peaks of pure HA. EDX results showed that the deposited films consisted of Ca, P, Ti, O and Ag, all of which distribute uniformly. With regard to the antibacterial effect, 98% of S. aureus and more than 99% of E. coli were killed after 24 h incubation and pictures of SEM showed obviously fewer cells on the surface with coating.     

Molecular imaging of in vivo calcium ion expression in area postrema of total sleep deprived rats: Implications for cardiovascular regulation by TOF-SIMS analysis

Excessive calcium influx in chemosensitive neurons of area postrema (AP) is detrimental for sympathetic activation and participates in the disruption of cardiovascular activities. Since total sleep deprivation (TSD) is a stressful condition known to harm the cardiovascular function, the present study is aimed to determine whether the in vivo calcium expression in AP would significantly alter following TSD by the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and calretinin (a specific calcium sensor protein in AP neurons) immunohistochemistry. The results indicated that in normal rats, the calcium intensity was estimated to be 0.5 × 10⁵ at m/z 40.08. However, following TSD, the intensity for calcium ions was greatly increased to 1.2 × 10⁵. Molecular imaging revealed that after TSD, various strongly expressed calcium signals were distributed throughout AP with clear identified profiles instead of randomly scattered within this region in normal rats. Immunohistochemical staining corresponded well with ionic image in which a majority of calcium-enriched gathering co-localized with calretinin positive neurons. The functional significance of TSD-induced calcium augmentation was demonstrated by increased heart rate and mean arterial pressure, clinical markers for cardiovascular dysfunction. Considering AP-mediated sympathetic activation is important for cardiovascular regulation, exaggerated calcium influx in AP would render this neurocircuitry more vulnerable to over-excitation, which might serve as the underlying mechanism for the development of TSD-relevant cardiovascular deficiency.     

Nanoscale characterization of the interface between bone and hydroxyapatite implants and the effect of silicon on bone apposition

Silicon plays an important role in bone mineralization and formation and is therefore incorporated into a wide variety of medical implants and bone grafts used today. The significance of silicon (Si) can be understood through an analysis of the mechanisms of bone bonding to calcium containing biomaterials and through comparisons of hydroxyapatite (HA) and silicon-substituted hydroxyapatite (Si–HA). The addition of Si to HA causes a decrease in grain size that subsequently affects surface topography, dissolution–reprecipitation rates and the bone apposition process. Through the use of high-resolution transmission electron microscopy (HR-TEM) studies, the interactions between bone and silicon hydroxyapatite (Si–HA) at interfaces are reviewed and related to their impact on bone apposition and ultimately the performance of medical implants.     

Surface bioactivity modification of titanium by CO2 plasma treatment and induction of hydroxyapatite: In vitro and in vivo studies

Since metallic biomaterials used for orthopedic and dental implants possess a paucity of reactive functional groups, bioactivity modification of these materials is challenging. In the present work, the titanium discs and rods were treated with carbon dioxide plasma and then incubated in a modified simulated body fluid 1.5SBF to obtain a hydroxyapatite layer. Surface hydrophilicity of samples, changes of surface chemistry, surface morphologies of samples, and structural analysis of formed hydroxyapatite were investigated by contact angle to water, X-ray photoelectron spectrometer (XPS), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and X-ray diffraction (XRD). The results demonstrated that hydrophilicity of titanium surface was improved and hydroxyl groups increased after modification with carbon dioxide plasma treatment. The hydroxyl groups on the surface of titanium were the richest after carbon dioxide plasma treatment under the condition of 20 W for less than 30 s. The hydroxyapatite formability of titanium surface was enhanced by carbon dioxide plasma pretreatment, which was attributed to the surface chemistry. MC3T3-E1 cell as a model cell was cultured on the Ti, CPT-Ti and CPT/SBF-Ti discs in vitro, and the results of the morphology and differentiation of the cell showed that CPT/SBF-Ti was the highest bioactive. The relative parameters of the new bone around the Ti and CPT/SBF-Ti rods including bone mineral density (BMD), a ratio of bone volume to total volume (BV/TV), trabecular thickness (Tb.Th.) and trabecular number (Tb.N.) were analyzed by a micro-computed tomography (micro-CT) after 4-, 8- and 12-week implantation periods in vivo. The results indicated that the CPT/SBF-Ti was more advantageous for new bone formation.     

Characteristic of microarc oxidized coatings on titanium alloy formed in electrolytes containing chelate complex and nano-HA

Microarc oxidized (MAO) TiO₂-based coatings containing Ca and P on titanium alloy were formed in electrolytes containing nano-hydroxyapatite (nano-HA), calcium and phosphate salts. The effects of HA concentration on the thickness, micropore size and number of the MAO coatings were not pronounced. However, the surfaces of the MAO coatings become rough and the crystallinity of anatase increases with increasing HA concentration. In addition, the Ca and P concentrations on the surfaces of the MAO coatings decrease, since the chelate complex of CaY²⁻ (Y = [₂(OOC)NCH₂CH₂N(COO)₂]⁴⁻) and phosphate ions are hindered to be incorporated into the MAO coatings by HA. In vitro experiments indicate that the apatite-forming abilities of the MAO coatings decrease with increasing HA concentration. Furthermore, with increasing HA concentration, the solubility of Ca and P of the MAO coatings decreases, which could lower the supersaturation of the SBF with respect to apatite near the surfaces of the MAO coatings, further leading to the decreased apatite-forming ability. The results indicate that the HA addition in the electrolytes has an important effect on the structure and in vitro bioactivity of the MAO coatings.     

Preparation and properties of titanium oxide film on NiTi alloy by micro-arc oxidation

Titanium oxide ceramic coatings were prepared by micro-arc oxidation (MAO) in galvanostatic regime on biomedical NiTi alloy in H₃PO₄ electrolyte using DC power supply. The surface of the coating exhibited a typical MAO porous and rough structure. The XPS analysis indicated that the coatings were mainly consisted of O, Ti, P, and a little amount of Ni, and the concentration of Ni was greatly reduced compared to that of the NiTi substrate. The TF-XRD analysis revealed that MAO coating was composed of amorphous titanium oxide. The coatings were tightly adhesive to the substrates with the bonding strength more than 45 MPa, which was suitable for medical applications. The curves of potentiodynamic porlarization indicated that the corrosion resistance of NiTi alloy was significantly improved due to titanium oxide formation on NiTi alloy by MAO.     

Fabrication of hydroxyapatite and TiO2 nanorods on microarc-oxidized titanium surface using hydrothermal treatment

AC-type microarc oxidation (MAO) and hydrothermal treatment techniques were used to enhance the bioactivity of commercially pure titanium (CP-Ti). The porous TiO₂ layer fabricated by the MAO treatment had a dominant anatase structure and contained Ca and P ions. The MAO-treated specimens were treated hydrothermally to form HAp crystallites on the titanium oxide layer in an alkaline aqueous solution (OH-solution) or phosphorous-containing alkaline solution (POH-solution). A small number of micro-sized hydroxyapatite (HAp) crystallites and a thin layer composed of nano-sized HAps were formed on the Ti-MAO-OH group treated hydrothermally in an OH-solution, whereas a large number of micro-sized HAp crystallites and dense anatase TiO₂ nanorods were formed on the Ti-MAO-POH group treated hydrothermally in a POH-solution. The layer of bone-like apatite that formed on the surface of the POH-treated sample after soaking in a modified simulated body fluid was thicker than that on the OH-treated samples.     

Effect of strontium ranelate on bone mineral: Analysis of nanoscale compositional changes

Strontium ranelate has been used to prevent bone loss and stimulate bone regeneration. Although strontium may integrate into the bone crystal lattice, the chemical and structural modifications of the bone when strontium interacts with the mineral phase are not completely understood. The objective of this study was to evaluate apatite from the mandibles of rats treated with strontium ranelate in the drinking water and compare its characteristics with those from untreated rats and synthetic apatites with and without strontium. Electron energy loss near edge structures from phosphorus, carbon, calcium and strontium were obtained by electron energy loss spectroscopy in a transmission electron microscope. The strontium signal was detected in the biological and synthetic samples containing strontium. The relative quantification of carbon by analyzing the C_K edge at an energy loss of ΔE = 284 eV showed an increase in the number of carbonate groups in the bone mineral of treated rats. A synthetic strontium-containing sample used as control did not exhibit a carbon signal. This study showed physicochemical modifications in the bone mineral at the nanoscale caused by the systemic administration of strontium ranelate.     

Effect of microgravity and a high magnetic field on hydroxyapatite deposition and implications for bone loss in space

The microgravity and high magnetic field simulated by a superconducting magnet were used to investigate the dynamics of deposition mechanisms of hydroxyapatite (HAp) on the surface of Ti-6Al-4V samples. The HAp coatings were derived by a self-assembly-induced biomineralization method in simulated body fluid. The experimental results showed that the amount and growth rate of HAp deposition in the simulated microgravity condition were far less than those in the gravity field, as were those in the high magnetic field compared with those outside the magnet. This may provide insight into the bone loss of astronauts in spaceflight from the viewpoint of the dynamic balance between HAp nucleation, deposition and decomposition on the surface of substrates.     

Effects of thermal annealing on titanium oxide films prepared by ion-assisted deposition

Titanium oxide thin films are prepared at a substrate temperature of 250 °C by electron-beam evaporation and ionassisted deposition. The effects of thermal annealing temperatures from 100 to 450 °C on the optical and mechanical properties are studied. The optical and mechanical properties include refractive indices, extinction coefficients, residual stress, surface roughness and crystallization. Experimental results show these properties of titanium oxide films clearly depend on the thermal annealing process.